In sewage 



Duff and associates (194.0) made extensive studies on the effect 

 of polluted water on the viability of B. salmonicida . Briefly, their 

 methods were as follows; experimental containers consisted of /^-liter 

 florence flasks, each containing 2 liters of fluid. Samples withdraim 

 for estimation of bacterial numbers were always at least 1 cc. in 

 volume, and were talcen after thorough mixing of the contents of the 

 container. Where necessary, decimal dilutions of such samples were 

 made, and from such dilutions triplicate platings were carried out in 

 most instances. In cases vihere large numbers of bacteria were present, 

 only those plates were used for count estimations which contained more 

 than 40 and fewer than 200 colonies per plate. Inocula consisted of 

 standardized suspensions invariably made from 2/4.-hour agar slant cultures 

 of B. salmonicida . 



A known volume of sample or of sample dilution (usually 1 cc. ) 

 was placed in the center of a previously poured and dried plate of 

 ordinary nutrient agar. By means of a sterile glass spreader, and by 

 rolling, the entire surface was uniformly wetted with the inoculum. 

 About 2 hours with tilted lid at 26° G. were allowed for absorption and 

 evaporation before finally inverting the plate. Incubation was carried 

 on at 22 i ° C. A differential medium was developed which served to 

 distinguish B. salmonicida from most sewage microflora. 



All sewage used in the experiments of Duff and associates (194-0) 

 was known to be free from trade and other chemical wastes. The actual 

 "sewage" placed in experimental flasks consisted of decanted supernatant 

 fluid from fresh sewage which had stood 2 hours in the laboratory. 



Undiluted Fresh Sewage 



Using an initial inoculum of 350 - 50 B. salmonicida per cc. Duff 

 and associates (1940) noted an increase in 24 hours of well over 2,000 

 per cc. The figure of "over 2,000 per cc." was a minimiim figure based 

 en having picked from two of three plates of a 1; 1,000 sample dilution, 

 two and four colonies, respectively, whose identity was subsequently 

 confirmed by morphological and cultural methods. Counts of similar 

 magnitude were obtained for 15 days, after which time increase of sewage 

 flora in experimental vessels rendered ftirther identification impossible. 



In another experiment the original bacterial content was partly 

 destroyed by heating at 56° C. for 2 hours. Sewage count was thus 

 reduced from 2,750 per cc. to about 130 per cc. The inoculum consisted 

 of 100 - 50 B. salmonicida per cc. Again a definite multiplication 

 followed, with a count of 200,000 B. salmonicida per cc. at the end of 

 the third day. Confirmed B^ salmonicida colonies were recovered up to 

 the twenty- third day. 



Plehn (1924) found that B. salmonicida in polluted water taken 

 from below Munich increased from 5,800 per cc. at the beginning of the 

 experiment to 8,803,000 per cc. at the end of 3 days. 



